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ENZYMES AND ENYZME KINETICS PART 1

Introduction

Enzymes are biological catalysts. These proteins are responsible for essentially all the biochemical reactions that occur in any form of life. Enzymes work by binding to molecules to speed up (catalyze) chemical reactions. The molecules that enzymes bind to and change are called the substrate. The three-dimensional shape of an enzyme determines its activity. Substrate molecules fit into a specific area of the enzyme molecule – called the active site. It is in the active site that the chemical reaction takes place, either combining the substrate molecule with other molecules, (an anabolic reaction) or splitting the substrate into subunits (a catabolic reaction). The resulting new molecule or subunits (called the products) detach from the enzyme molecule, allowing the enzyme to catalyze another reaction.

The three-dimensional shape of the enzyme is crucial to its function. Other chemicals can bind to an enzyme and modify its shape. Sometimes these other chemical molecules are necessary to adjust the shape of the enzyme to fit the substrate. These molecules are called cofactors and are required for some enzymes to be active. Many of our essential minerals and vitamins function as cofactors.

Other kinds of molecules change the shape of enzymes so that they cannot function. These kinds of molecules are called inhibitors. Inhibitors prevent enzymes from working in two different ways. Noncompetitive inhibitors bind to enzymes and change the shape of the enzyme’s active site so that the substrate cannot fit. Competitive inhibitors are molecules that fit onto the active site of the enzyme, but do not react. Because there is limited number of enzyme molecules, these molecules would compete with the normal substrate for the active sites. There would then be fewer enzymes available for the substrate, causing the reaction to proceed more slowly.

Procedure – work in groups of 4

Use the playdough at your table to design the following enzymes and substrate molecules. (Use different colors for your enzyme and substrate molecules.) Be sure to include an active site that is specific to your substrate molecule. Be prepared to describe your design to your classmates and your instructor.

1. Design an enzyme and substrate. Indicate whether your enzyme is catalyzing a catabolic reaction or an anabolic reaction.

2. Design an enzyme that requires a cofactor to function. Be sure to include your substrate molecules.

3. Design an enzyme with a non-competitive inhibitor. Be sure to include your substrate molecules.

4. Design an enzyme with a competitive inhibitor. Be sure to include your substrate molecules.

Experiment #1

In this experiment, you will study catalase found in liver cells. Catalase is found in the cells of many living tissues. It speeds up the reaction that breaks down hydrogen peroxide (H 2 O 2 ), a toxic chemical, into 2 nontoxic substances-water and oxygen gas. The reaction is as follows:

2 H 2 O 2 2H 2 O + O 2

Hydrogen peroxide is produced as a byproduct of many normal cellular reactions. High levels of hydrogen peroxide, however, can damage the cell. Catalase prevents hydrogen peroxide from building up to dangerous levels in cells.

Question We will be using chicken livers as a source of catalase. How will we know if the enzyme in the liver is still viable?

Hypothesis Construct a hypothesis based on the above question that is testable.

Prediction Predict the result of the following experiment based on your hypothesis. If the enzyme is still active and you put some liver in hydrogen peroxide, what would you expect to see and why?

Procedure

Note: Make sure to clean your stirring rod (and your test tube if necessary) between steps for better results. Be sure to record your observations before continuing with the next step in the experiment.

1. Place 2 ml of 3% hydrogen peroxide solution into a clean test tube. Repeat with a second test tube. Label them tube A and tube B.

2. Cut a small piece of liver (use forceps and scissors), and add it to the test tube B. Use the stirring rod to push it into the tube that contains the hydrogen peroxide solution.

3. Make up a simple chart in your lab notebook and record your observations. Do you see a reaction? What is the purpose of tube A? Explain your results in terms of your hypothesis.

Experiment #2

Question

What causes an enzymatic reaction to slow down or stop? Can the enzyme get used up? Can the substrate get used up?

Hypothesis

Construct a hypothesis based on the above question that is testable.

Prediction

Predict the result of the following experiment based on your hypothesis.

After the reaction stops, will fresh liver added to the “used” substrate react with it? Explain why or why not.

Will fresh substrate added to the “used” liver react with it? Explain why or why not.

Procedure

1. Pour 2 ml of 3% hydrogen peroxide solution into a clean test tube labeled tube #1 and add a small piece of liver. Check the rate of reaction for 20 minutes or until the reaction stops or slows considerably.

2. Decant the liquid (but not the liver) from tube #1 into a clean test tube labeled tube #2. What is this liquid composed of?

3. Add a fresh piece of liver to the liquid in tube #2. What happened? Record your results and explain your results in terms of your hypothesis.

4. Pour 2 ml of fresh 3% hydrogen peroxide solution to the liver remaining in the first tube. Can you observe any reaction? Record your results and explain your results in terms of your hypothesis.

Experiment #3

Inhibitors of Catalase Hydroxylamine is an inhibitor of catalase. Hydroxylamine attaches to the catalase molecules and interferes with the formation of normal enzyme substrate complex.

Question Is hydroxylamine a competitive or noncompetitive inhibitor?

Hypothesis Construct a hypothesis based on the above question that is testable.

Prediction Predict the result of the following experiment based on your hypothesis.

How will adding additional substrate affect the reaction if hydroxylamine is a competitive

inhibitor?

if

hydroxylamine is a noncompetitive inhibitor?

Procedure

Caution! Hydroxylamine is toxic--handle with care and wash your hands after the experiment. Notify your instructor immediately if a spill occurs.

1. Pour 2 ml of hydrogen peroxide into a clean test tube labeled tube C. Repeat with a second test tube and label it tube D.

2. Add 5 drops of 5% hydroxylamine solution to tube D.

3. Use forceps and scissors to cut two small pieces of liver of approximately the same size. Add one piece of liver to each test tube and observe the reactions. Do you see a difference in the rate of the reactions? Make up a simple chart and record your observations.

4. Add an additional 2 ml of hydrogen peroxide to tube D and compare the rate of reaction to tube C. Do you see any changes in reaction rate? Add another 2 ml of hydrogen peroxide. Do you see any changes in reaction rate? What do your results tell you about the nature of hydroxylamine inhibition?

Once you have finished with this introduction to enzymes, continue to the next page to start Enzyme Kinetics part 1 using a new enzyme system.

Enzyme Kinetics, part I

Previously we studied the nature of enzyme activity using catalase in liver tissue. We were able to determine under what conditions the enzyme worked or not. Now, however, we wish to examine how the rate of enzyme-catalyzed reactions varies. Enzyme reaction rates depend on physical parameters such as substrate concentration, pH, temperature, and other factors such as the chemical nature of the substrate and catalyst. We will examine these characteristics using the enzyme tyrosinase (also called catechol oxidase).

Tyrosinase is found in many plants and animals where it is responsible for a biochemical pathway beginning with the amino acid, tyrosine (hence the name “tyrosinase”) and leading ultimately to the formation of melanin, a black pigment. Specifically the enzyme converts tyrosine to Dopa and then converts Dopa to Dopaquinone. Activity of this enzyme in part accounts for the darkening of human skin in response to sunlight and the darkening of freshly peeled potatoes and other fruits and vegetables when exposed to air. A temperature sensitive mutation in cats is what causes the unusual pigmentation in Siamese cats- the pigment is only synthesized in cooler tissues. Albinism results when the enzyme is defective, and there are many mutations linked with the tyrosinase gene. (For more information on albinism visit the NOAH website at: http://www.albinism.org/ or the research lab page of Dr. King at http://albinism.med.umn.edu/) (figure from the King web page site)

) (figure from the King web page site) Tyrosinase will react with a variety of substrates.

Tyrosinase will react with a variety of substrates. This can include its natural substrate, tyrosine, and catechol, a phenol also found in plant cells. In the experiments below, catechol is used as a substrate for tyrosinase. In the presence of oxygen, tyrosinase converts catechol into benzoquinone, a pigmented product. Thus the product of the enzyme reaction is visible and we can use the intensity of color formation to quantify the enzyme reactions - more color means more product is produced. We can easily measure this using a spectrophotometer. Recording the amount of product produced over time will allow us calculate the rate of enzyme activity.

Methods

Preparation of Enzyme

1. Peel and wash a medium-sized potato. Chop the cleaned potato into small pieces and place in a blender with 40 mL ice-cold distilled water. Blend at highest speed for one minute. Filter through two large layers of cheesecloth into two 50 mL chilled centrifuge tubes.

2. Centrifuge at 500xg for five minutes. Remove 10 mL of the supernatant from each tube and place in a chilled test tube labeled “Supernatant”. Keep on ice.

3. Makeup 40mL of a10% solution of the supernatant with ice-cold distilled water and place in a 125 mL Erlenmeyer flask labeled “Enzyme”. Keep on ice.

Using the spectrophotometer to assay tyrosinase.

The spectrophotometer is used to measure the concentration of compounds or particles in a solution based on the amount of light that passes through the solution. The choice of wavelength for measuring a particular substance is based on the color of light that is absorbed by that substance.

In order to make accurate measurements with a spectrophotometer, you must go through the following steps.

1. Allow the spectrophotometer to warm-up for at least 10 minutes. Turn it on before you get your test tubes ready. Set wavelength at 540 nm.

2. Mix all ingredients in your test tubes except for the enzyme. You want to use a solution that contains everything except the substance you want to measure (a so- called "blank") to set the baseline absorbance to 0. Because there is no enzyme present, no product will be produced. By setting absorbance to 0 for the blank, only the newly produced material will be sensed by the machine when you do a real experiment with enzyme present. Make sure the tube is covered and adjust the spectrophotometer to read zero absorbance.

3. You must minimize extraneous influences on light readings. To do this, you should take the following precautions: mark the tubes so that they are always read in the same orientation, Use a Kimwipe to clean condensation and fingerprints off the outside of the tubes before placing them in the spectrophotometer. These steps will reduce variation due to unevenness of the glass or dirt on the tubes. Always cover the sample chamber when taking a reading.

4. Start the reactions by adding the enzyme to your tube, mixing quickly, cleaning the outside of the tube and inserting the tube into the spectrophotometer. You will only have about 20 seconds to do this. Immediately record the absorbance of your tube. Keep the rest of your enzyme on ice when not in use.

Remember, when the enzyme and substrate are together, the reaction has begun!

5. Remember your starting time. Read the absorbance (Abs) every 30 seconds for 5 minutes or until the absorbance readings remain the same for three readings in a row.

6. Remove the tube and start a new reaction. Most reactions will be over in about 3-4 minutes. You will have enough test tubes to set up a whole series of reactions in advance if you wish.

Experiments

Experiment #1 - Activity of tyrosinase in potato extract

Questions

Your instructor has prepared fresh potato extract to test for tyrosinase. How will you know that the tyrosinase is active?

Since catechol is present in some plant tissues, how will you know that the potato extract does not contain catechol?

Hypotheses Construct hypotheses based on the above questions that are testable.

Predictions Predict the results of the following experiments based on your hypotheses.

Procedure

1. Use the following table to run three experimental test tubes. Remember to add the enzyme last - just before you are ready to run your experiment.

Tube #

Buffer, pH 6.0 0.1M, NaPO 4 ,

0.006 M catechol,ml

H

2 O,ml

Enzyme, ml

ml

A

1.0

ml

0.0

ml

2.0

ml

1.0

ml

B

1.0

ml

1.0

ml

2.0

ml

0.0

ml

C

1.0

ml

1.0

ml

1.0

ml

1.0

ml

2. Start the reactions by adding the enzyme to your tube, mixing quickly, cleaning the outside of the tube and inserting the tube into the spectrophotometer. You will only have about 20 seconds to do this. Immediately record the absorbance of your tube. Keep the rest of your enzyme on ice when not in use.

Remember, when the enzyme and substrate are together, the reaction has begun!

3. Remember your starting time. Read the absorbance (Abs) every 30 seconds for 5 minutes or until the absorbance readings remain the same for three readings in a row.

4. Remove the tube and start a new reaction.

5.

Construct a data table to record your results. What is the purpose of each experimental tube? Explain your results based on your hypothesis.

6. Construct a graph to illustrate your results from Tube C. Plot absorbance on the y-axis and time on the x-axis. Make sure you indicate your units and label your graph.

7. Analyze your graph. Do the absorbance readings change steadily over time? Do the readings level off at some point? Explain your results.

8. Label the part of your graph where you think the absorbance readings are changing the fastest. Label the part of your graph where you think the absorbance readings are changing the most slowly. What do these parts of the curve look like?

9. Reanalyze your results by calculating the rate of tyrosinase reaction for each 1-minute interval.

10. Graph your rates of reaction as a function of time. Your rates should be on the y-axis and time should be on the x-axis. Be sure to indicate your units and label your graph.

11. What do you conclude from this experiment? Do all time intervals provide the same reaction rate? Why or why not? During what time interval do you get your highest reaction rate?